Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66.
The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparation behaved homogeneously on gel filtration on Sephadex G-150 in aqueous Triton X-100 and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 55,000. The enzyme requires Mn2+, and only UDP-GalNAc served as a sugar donor. Apomucin, A1 protein, kappa-casein, apofetuin, and apoantifreeze glycoproteins served as acceptors, but the rate and amount of the transfer varied considerably from one acceptor to another. The transfer reaction terminated at the level of glycosylation of from only a few to at most about 40% of the serine plus threonine residues from which mucin-type oligosaccharides had been removed. This indicates that the transferase requires a certain conformation surrounding the acceptor site, but suggests also that a special mechanism may be functioning in vivo for frequent glycosylation of the abundant serine plus threonine residues of mucins. Lacto-N-fucopentaose I, ceramide di- and trihexosides, and globoside were not acceptors.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005700:Galactosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D017350:N-Acetylgalactosaminyltransferases; D051381:Rats; D013379:Substrate Specificity; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D005700:Galactosyltransferases; D008345:Manganese; D017350:N-Acetylgalactosaminyltransferases; D014536:Uridine Diphosphate N-Acetylgalactosamine; D000097763:Polypeptide N-acetylgalactosaminyltransferase